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Figure S1. Demonstration of HCV replication by HCV-dependent fluorescent reporter translocation and GLuc secretion. (A), HCV-dependent fluorescent reporter translocation in HFLC infected with HCVcc Clone 2 or with Jc1FLAG2(ns-Gluc2a). HFLC were transduced with TRIP SV40/Alb tagRFP nlsIPS-1 pseudoparticles. Cells were cultured for 48-96 hours to permit expression of the reporter, then infected with either Clone 2 or Jc1G, with or without added 2′CMA or Pyridone-6, as detailed in Materials and Methods. Cells were photographed on a Nikon Eclipse TE300 fluorescent microscope (Nikon Instruments, Inc., Melville, NY, USA) using a Spot RT Color Camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) at 48 hours after infection. Magnification, 20X. Data are representative of three independent experiments. (B), HCV-dependent fluorescent reporter does not translocate in mock-infected HFLC. (C), GLuc secreted by HFLC following infection with Jc1G. Culture supernatants were sampled for GLuc measurement at the indicated times after infection. Values of P (comparing Jc1G vs. UV-treated Jc1G or Jc1G + 2′CMA) were determined by repeated measures ANOVA, and are indicated on each graph.

Figure S2. Kinetics of changes in gene expression in HFLC infected with either HCVcc Clone 2 or Jc1G. HFLC cultures from two donors were each infected with either Clone 2 or Jc1G, or left in culture uninfected. At each time point shown, cells were harvested for RNA preparation and the RNA subjected to real-time RT-PCR for evaluation of gene expression. At each time point, the level of each mRNA in the infected wells was compared to that in uninfected wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of two measurements per well, two wells per point. Data are from 4 experiments and are representative of 6 experiments with comparable results. (A), Cytokines and chemokines. (B), ISGs. (C), Signal transduction genes.

Figure S2. Kinetics of changes in gene expression in HFLC infected with either HCVcc Clone 2 or Jc1G. HFLC cultures from two donors were each infected with either Clone 2 or Jc1G, or left in culture uninfected. At each time point shown, cells were harvested for RNA preparation and the RNA subjected to real-time RT-PCR for evaluation of gene expression. At each time point, the level of each mRNA in the infected wells was compared to that in uninfected wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of two measurements per well, two wells per point. Data are from 4 experiments and are representative of 6 experiments with comparable results. (A), Cytokines and chemokines. (B), ISGs. (C), Signal transduction genes.

Figure S2. Kinetics of changes in gene expression in HFLC infected with either HCVcc Clone 2 or Jc1G. HFLC cultures from two donors were each infected with either Clone 2 or Jc1G, or left in culture uninfected. At each time point shown, cells were harvested for RNA preparation and the RNA subjected to real-time RT-PCR for evaluation of gene expression. At each time point, the level of each mRNA in the infected wells was compared to that in uninfected wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of two measurements per well, two wells per point. Data are from 4 experiments and are representative of 6 experiments with comparable results. (A), Cytokines and chemokines. (B), ISGs. (C), Signal transduction genes.

Figure S3. Effects of exogenous poly I:C, IFNβ, or IL29 on gene expression in HFLC cultured without virus. HFLC cultures from two donors were each cultured in the presence of no stimulus, 5 μg/ml p(I:C), 1 U/ml recombinant human IFNβ, or 10 ng/ml recombinant human IL29 as indicated. At the indicated times, cells were harvested for RNA preparation and the RNA subjected to real-time RT-PCR for evaluation of gene expression. At each time point, the level of each mRNA in the treated wells was compared to that in untreated wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of measurements from two wells. Similar data were obtained in three other experiments. (A), Expression of cytokine and chemokine genes. (B), Expression of ISGs.

Figure S3. Effects of exogenous poly I:C, IFNβ, or IL29 on gene expression in HFLC cultured without virus. HFLC cultures from two donors were each cultured in the presence of no stimulus, 5 μg/ml p(I:C), 1 U/ml recombinant human IFNβ, or 10 ng/ml recombinant human IL29 as indicated. At the indicated times, cells were harvested for RNA preparation and the RNA subjected to real-time RT-PCR for evaluation of gene expression. At each time point, the level of each mRNA in the treated wells was compared to that in untreated wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of measurements from two wells. Similar data were obtained in three other experiments. (A), Expression of cytokine and chemokine genes. (B), Expression of ISGs.

Figure S4. Gene expression in cultures of conventional vs. gradient-enriched HFLC stimulated with virus and/or p(I:C). HFLC (Organ AB-021011) were plated as for other experiments or were enriched by density gradient centrifugation before plating. HFLC were infected with Clone 2 or left uninfected. At each time point, the level of each mRNA in the infected wells was compared to that in uninfected wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of two measurements per well, two wells per point. (A), Cellular RNA was harvested at the indicated times for measurement of HCV RNA and cellular mRNA. (B), At 90 hours after the start of infection, cells were treated with 5 ?g/ml p(I:C) or with an equal volume of PBS. Six hours later, cells were harvested for measurement of mRNA. *, p < 0.05. **, p < 0.01 (1-way ANOVA with Bonferroni multiple comparison post-test). Data are representative of three experiments (livers from three separate donors) with comparable results.

Figure S4. Gene expression in cultures of conventional vs. gradient-enriched HFLC stimulated with virus and/or p(I:C). HFLC (Organ AB-021011) were plated as for other experiments or were enriched by density gradient centrifugation before plating. HFLC were infected with Clone 2 or left uninfected. At each time point, the level of each mRNA in the infected wells was compared to that in uninfected wells from the same HFLC prep at the same time point. Each point shows the mean ± SD of two measurements per well, two wells per point. (A), Cellular RNA was harvested at the indicated times for measurement of HCV RNA and cellular mRNA. (B), At 90 hours after the start of infection, cells were treated with 5 ?g/ml p(I:C) or with an equal volume of PBS. Six hours later, cells were harvested for measurement of mRNA. *, p < 0.05. **, p < 0.01 (1-way ANOVA with Bonferroni multiple comparison post-test). Data are representative of three experiments (livers from three separate donors) with comparable results.

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